Integrins are members of a family of heterodimeric transmembrane cell surface receptors that play a crucial role in cell-cell and cell-matrix adhesion processes (Hynes, R. O. Cell 1992, 69, 11-25). These receptors consist of an α- and a β-subunit, which non-covalently associate in defined combinations (Eble, J. A. Integrin-Ligand Interaction; Springer: Heidelberg, 1997; pp 1-40). Most of them recognize the Arg-Gly-Asp (RGD) triad found in many extracellular matrix proteins (i.e vitronectin) (Serini, G.; et al. A sticky business. Exp. Cell. Res. 2005, in press) and snake venom disintegrins (Ruoslahti, E.; Pierschbacher, M. Cell 1986, 44, 517-518; D'Souza, S. E.; et al. Trends Biochem. Sci. 1991, 16, 246-250; Gould, R. J.; et al. Proc. Soc. Exp. Biol. Med. 1990, 195, 168-171). Even if different integrins recognize different proteins containing the RGD sequence, several studies have demonstrated that the amino acid residues flanking the RGD sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes. Despite numerous studies reported in the literature, ligand selectivity toward different integrin subtypes is still a challenging problem mainly because most of the 3D-structures of integrin subtypes remain unknown (Marinelli, L.; et al. J. Med. Chem. 2004, 47, 4166-4177).
An extensively studied member of this receptor class is integrin αvβ3. This integrin is strongly expressed on activated endothelial and melanoma cells, in contrast, it is weakly expressed in quiescent blood vessels and pre-neoplastic melanomas (Hood, J. D.; Cheresh, D. A. Nat. Rev. Cancer 2002, 2, 91-100). Along with αvβ5 integrin, αvβ3 is reported to be involved in physiological processes including angiogenesis and tissue repair as well as pathological conditions such as tumor induced angiogenesis (Eliceiri, B. P.; Cheresh, D. A. J. Clin. Invest. 1999, 103, 1227-1230; Kumar, C. C. Curr. Drug Targets 2003, 4, 123-131), tumor cell migration and invasion (Clezardin, P. Cell. Mol. Life. Sci. 1998, 54, 541-548). Despite the fact that both integrins promote cell attachment to vitronectin and participate in the same processes, they are reported to be structurally designed to respond to different signaling events. Previous studies provided evidence that bFGF-induced angiogenesis is mediated by αvβ3 whereas VEGF-induced angiogenesis is mediated by αvβ5 (Friedlander, M.; et al. Science 1995, 270, 1500-1502). Melanoma cells expressing αvβ3 migrate in vitro and metastasize in vivo without the need for exogenous cytokine stimulation (Filardo, E. J.; et al. J Cell Biol. 1995, 130, 441-450). Conversely, tumor cells expressing αvβ5 integrin require a tyrosine kinase receptor-mediated signaling event for motility on vitronectin and in vivo dissemination (Brooks, P. C.; et al J Clin Invest. 1997, 99, 1390-1398). While αvβ5 is widely expressed by many malignant tumor cells, αvβ3 has a relatively limited cellular distribution compared with that of αvβ5 (Pasqualini, R.; et al. J. Cell Science 1993, 105, 101-111; Walton, H. L.; et al. J. Cell. Biochem. 2000, 78, 674-680.). Therefore, in order to target αvβ3-mediated processes for diagnostic or therapeutic purposes, the development of new compounds that can discriminate between αvβ3 and αvβ5 is required.
To date, various therapeutic candidates, including antibodies (Gutheil, J. C. et al. Clin. Cancer Res. 2000, 6, 3056-3061), small molecules (Miller, W. H.; et al. Drug Discov. Today 2000, 5, 397-408; Marugan, J. J.; et al. J. Med. Chem. 2005, 48, 926-934), peptidomimetics (Sulyok, G. A.; et al. J. Med. Chem. 2001, 44, 1938-1950; Belvisi, L.; et al. Org. Lett. 2001, 3, 1001-1004), and cyclic peptides (Mitjans, F. et al. Int. J. Cancer 2000, 87, 716-723; Dechantsreiter, M. A.; et al J. Med. Chem. 1999, 42, 3033-3040) have been clinically evaluated and shown to successfully modulate αvβ3-mediated processes. So far, the pentapeptide cyclo (-Arg-Gly-Asp-D-Phe-NMeVal-), referred to as c(RGDf[NMe]V) (Eskens, F. A.; et al Eur. J. Cancer 2003, 39, 917-926), is one of the most active αvβ3 antagonists reported in the literature. Previous studies have demonstrated a higher affinity of this peptide for integrin αvβ3 as compared to αvβ5 and have reported inhibition of αvβ3-mediated cell adhesion with IC50 values in the micromolar range when assayed in different tumor cell lines (Goodman, S. L.; et al. J. Med. Chem. 2002, 45, 1045-1051).
The crystal structures of the extracellular segment of integrin αvβ3 in its unligated state and in complex with c(RGDf[NMe]V) and the docking studies on αvβ3 integrin ligands have shown that the main interactions are between the positively charged arginine and the α-subunit and between the anionic aspartic acid and the β-subunit (Marinelli, L.; et al. J. Med. Chem. 2003, 46, 4393-404; Xiong, J. P. et al. Crystal structure of the extracellular segment of integrin αvβ3. Science 2001, 294, 339-345; Xiong, J. P.; et al. Science 2002, 296, 151-155), and that selectivity between different subunits is achieved by the RGD sequence conformation. Previous studies also reported that echistatin, the smallest (49 residues) of the viper (Echis carinatus) disintegrins, is a potent antagonist of the integrins αvβ3, α5β1 and αIIbβ3 (Wierzbicka-Patynowski, I.; et al. J. Biol. Chem. 1999, 274, 37809-37814) and that the amino acids adjacent to the RGD motif together with the 41-49 C-terminal residues appear to be critical for the selective recognition of integrins. Mutation and photoaffinity cross-linking experiments, and NMR conformational analysis combined with docking studies Yahalom, D.; et al. Biochemistry 2002, 41, 8321-8331; Saudek, V.; et al. Biochemistry 1991, 30, 7369-7372), have provided evidence that the C-terminal region of echistatin binds to a site within the β3 subunit of the αvβ3 receptor, which is distinct from the sites that bind residues flanking the RGD triad in small peptide ligands.